WNTA5-mediated miR-374a-5p regulates vascular smooth muscle cell phenotype transformation and M1 macrophage polarization impacting intracranial aneurysm progression

miR-374a-5p expression and localization in intracranial aneurysm (IA) tissues were detected, and its correlation with vascular smooth muscle cells (VSMCs) and macrophage markers was analyzed. Using platelet-derived growth factor-BB (PDGF-BB) induced VSMC model, elastase-induced IA rat model. Subsequently, miR-374a-5p was knocked down or overexpressed. We investigated the effects of miR-374a-5p on phenotypic conversion, and in vivo experiments were also carried out to verify the findings. The targeted relationship between miR-374a-5p and WNTA5 was analyzed. The effect of WNT5A inhibition on VSMC phenotypic transformation and THP-1-derived macrophage polarization was explored. Clinical studies have shown that miR-374a-5p was upregulated in IA patients. miR-374a-5p was negatively correlated with SM22α, α-SMA, CD206, and positively correlated with CD86. In vitro experiments showed that knocking down miR-374a-5p reversed the promotion of SM22α and α-SMA expression by PDGF-BB, while overexpression of miR-374a-5p had the opposite effect. In addition, knocking down miR-374a-5p also reversed the decrease in Calponin, TIMP3, TIMP4, and IL-10 levels caused by PDGF-BB, and further reduced the levels of MMP1, MMP3, MMP9, IL-1β, IL-6, and TNF-α. These findings were further validated in vivo. In IA rats, there were notable increases in both systolic and diastolic blood pressure, along with an elevated M1/M2 ratio and the occurrence of vascular lesions. However, these symptoms were improved after knocking down miR-374a-5p. Furthermore, miR-374a-5p could target the WNT signals (WNT2B, WNT3, and WNT5A). miR-374a-5p regulated the VSMC phenotypic conversion and M1 macrophage polarization by targeting WNT5A, thereby impacting the progression of IA.

Intracranial aneurysm (IA) represents the primary trigger of subarachnoid hemorrhage, a condition linked to high morbidity and mortality 1 .The exact mechanisms responsible for the development and rupture of IA are still not entirely comprehended.However, it is widely believed that endothelial dysfunction, the phenotypic transformation of vascular smooth muscle cells (VSMCs), secretion of matrix metalloproteinases, and activation of innate immune cells contribute to the pathological changes observed in the vascular system 2 .Multiple studies have shown that VSMC phenotypic transformation serves as essential for the formation and progression of IA, and modulation of VSMC phenotypic transformation can attenuate the formation of IA 3,4 .In addition, macrophage polarization is implicated in the formation and rupture of IA 2 .It has been demonstrated that an M1-polarized macrophage increases the risk of IA rupture, while polarization towards an M2 phenotype can prevent IA rupture 5,6 .
MicroRNAs (miRNAs), as a class of endogenous non-coding RNAs, play a crucial role in the regulation of gene expression.They have been identified as potentially valuable biomarkers for disease diagnosis, prognosis, and therapeutic interventions 7,8 .Currently, there is evidence suggesting biological connections between miRNAs

Animals and treatment
Male Sprague-Dawley rats aged 8 weeks and bred under SPF conditions were selected from Hunan SJA Experimental Animal Co., Ltd, and divided randomly into four groups (n = 10 per group): Sham, IA, IA+ inhibitor NC, and IA+ miR-374a-5p inhibitor groups.Rats in the Sham group underwent sham surgery and were injected stereotactically with 1 μL PBS once per day.In the IA group, rats received stereotactic injection of 1 μL of a mixture of PBS and Lipofectamine 2000 once daily.In the IA+ inhibitor NC group, rats received a stereotactic injection of 1 μL of a mixture of inhibitor NC (4.5 μM) and Lipofectamine 2000 once a day.In the IA+ miR-374a-5p inhibitor group, rats received a stereotactic injection of 100 μL of a mixture of miR-374a-5p inhibitor (4.5 μM) and Lipofectamine 2000 once a day.The IA model was induced by elastase (E1250, Sigma-Aldrich) in the other groups.In brief, the rats were anesthetized with 3% isoflurane and positioned supinely, following which the right carotid artery was ligated with a 4-0 nylon suture.One week later, a small single hole was drilled, and 10 μL of elastase was injected into the right basal cistern according to the stereotactic coordinates 3,23 .A high salt diet (8% NaCl and 0.12% β-aminopropionitrile 23 ) was provided to induce hypertension in rats for 30 days.Measurements for baseline systolic blood pressure (SBP) and diastolic blood pressure (DBP) were taken at different time points: weeks 0, 1, 2, 3, and 4, after IA induction.After 30 days, rats were euthanized with CO 2 overdose.Subsequently, a subset of rats from each group (n = 3 per group) underwent perfusion fixation using PBS and 4% paraformaldehyde (PFA), followed by treatment with 2% India ink 4 .The perfused and treated samples were then used for subsequent tissue pathology analysis.The animal study was conducted with the approval of the ethics review committee of The Affiliated Changsha Hospital of Xiangya School of Medicine, Central South University (2022-7).All methods are implemented in accordance with ARRIVE guidelines and regulations.

Flow cytometry
For the detection of M1 and M2 macrophage infiltration, flow cytometry was performed.The cells were first rinsed with 1 mL of PBS after 1 × 10 5 cells (100 µL) were added in a 1.5 mL EP tube, followed by centrifugation at 1500 rpm for 5 min to obtain the sediment.After resuspending the cells in 100 µL of culture medium, CD86+ and CD206+ antibodies were added and incubated in the dark under room temperature for 30 min.The cell pellet, after washing with PBS and undergoing centrifugation, was ultimately suspended in 150 µL of basal medium for flow cytometry analysis.

Quantitative real-time PCR (qRT-PCR)
In this study, the TRIzol total RNA extraction kit (15596026, Thermo) was used to extract total RNA from the cells and tissues, followed by reverse transcription of the total mRNA into cDNA with the cDNA reverse transcription kit (CW2569, CWBIO).The primers for the target gene were designed using the Primer5 software, based on the sequence obtained from NCBI and synthesized by Beijing Tsingke.For relative gene expression analysis, the Ultra SYBR Mixture (CW2601, CWBIO) was used on a fluorescence quantitative PCR machine (QuantStudio1, Thermo).H-GAPDH, H-U6 or R-5S were utilized as internal reference genes to obtain the relative levels of the target gene, calculated using the 2 −ΔΔCt method with the help of their respective standard curves.Table 1 shows the primer sequences employed during the study.

Western blot (WB)
To extract proteins from tissues and cells, RIPA lysis buffer was utilized, followed by protein quantification using the BCA protein assay kit.Electrophoresis-separated protein lysates were transferred to nitrocellulose membranes, which were then blocked using 5% skim milk at room temperature for 90 min.Primary antibodies such as Calponin, TIMP3, TIMP4, SM22α, α-SMA, MMP1, MMP3, MMP9, WNT2B, WNT3, and WNT5A were used with GAPDA serving as the internal control.The membranes were left overnight in primary antibodies at 4 ℃, followed by washing with PBST.Secondary antibodies (HRP goat anti-mouse IgG and HRP goat antirabbit IgG) were diluted and left to incubate on the membrane for 90 min at room temperature.Following three PBST washes, the membrane was visualized and analyzed for grayscale using Quantity One software (Fig. S1).Antibody information is shown in Table 2.

Haematoxylin-eosin (HE) staining
Fixed tissue samples were embedded in paraffin and sectioned to 6 μm thickness.After treatment with xylene and dehydration using different concentrations of ethanol (100%, 95%, 85%, and 75%) for 5 min at each level, the sections were subjected to hematoxylin staining and eosin staining for 1 min, respectively.Gradual alcohol dehydration (95-100%) was then performed for 5 min at each level.Finally, the sections were washed with xylene twice for 10 min each and sealed with neutral gum for microscopic observation.
The co-localization of miR-374A-5p with SM22α and CD68 was detected by fluorescence microscope.

Dual-luciferase reporter assay
Initially, the binding site of miR-374a-5p on WNT5A was predicted using the TargetScan website (http:// www.targe tscan.org/ vert_ 71/), followed by amplifying the fragment.Next, the amplified fragment was inserted into the psiCHECK-2 vector to create a WNT5A wild-type (WT) plasmid.To construct the WNT5A mutant (MUT) plasmid, gene site mutagenesis technology was deployed to mutate some of the nucleotides in the binding site fragment.The constructed WNT5A gene dual luciferase reporter plasmid was co-transfected with either miR-374a-5p mimic or miR-374a-5p NC into 293 T cells (HG-NC071, HonorGene).Each group was subjected to luciferase activity detection, following the manufacturer's guidelines for the Dual-Luciferase Report Gene Assay Kit (E1910, Promega).

Ethical approval statement
The study was conducted with the approval of the ethics review committee of The Affiliated Changsha Hospital of Xiangya School of Medicine, Central South University (2022-7).All methods were performed in accordance with the relevant guidelines and regulations.

Statistical analysis
The experiments were conducted a minimum of three times.GraphPad Prism 8.0 software was utilized for data analysis, and the results were displayed as means ± standard deviation for all quantitative data.Initially, normality and homogeneity of variance tests were conducted, revealing equal variances and normal data distribution.Unpaired t-tests were implemented for between-group comparisons, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups.Results were marked statistically significant when P < 0.05.

miR-374a-5p was upregulated in IA patients and affected VSMC phenotypic transformation
Firstly, we detected the expression of miR-374a-5p, SM22α, α-SMA, CD86 and CD206 in human IA and normal tissues.Compared with the Normal group, the levels of miR-374a-5p and CD86 in the IA group were significantly increased, and SM22α, α-SMA and CD206 were significantly decreased (Fig. S2A).To further evaluate the cell distribution of miR-374a-5b in the arterial wall, we performed FISH-IF with the macrophage marker CD68 and VSMC marker SM22-α, respectively.As shown in Fig. S2B, miR-374a-5b (red) and CD68/SM22-α (green) were co-localized in the arteries.Compared with the Normal group, the proportion of miR-374a-5p + SM22-α + positive cells was relatively high.Pearson correlation analysis showed that the expression of miR-374a-5p was negatively correlated with the expression of SM22α, α-SMA, and CD206, and positively correlated with the expression of CD86 (Fig. S2C).To examine the role of miR-374a-5p in VSMC phenotypic transformation, we employed a PDGF-BB-induced VSMC model, specifically to gauge the impact of miR-374a-5p on the phenomenon.The upregulation of miR-374a-5p expression in PDGF-BB-induced VSMCs was successfully suppressed using miR-374a-5p inhibitor, as depicted in Fig. 1A.Utilizing IF double-staining to observe the expression of a contractile marker in VSMC cells, a decline in SM22α and α-SMA expression in the Model group was detected against the Control group, but an increase in the miR-374a-5p inhibitor group compared to the Model group (Fig. 1B).Moreover, WB outcomes demonstrated a reduction in SM22α, α-SMA, Calponin, TIMP3, and TIMP4 expressions induced by PDGF-BB treatment, while MMP1, MMP3, and MMP9 expressions increased.Nonetheless, miR-374a-5p inhibitor reversed these protein levels, proven by the enhanced expression (Fig. 1C).In cell supernatants, ELISA analysis highlighted higher levels of IL-1β, IL-6, and TNF-α and decreased IL-10 levels in the Model group relative to the Control group.However, the miR-374a-5p inhibitor group displayed the opposite trend against the Model group (Fig. 1D).In contrast, miR-374a-5p mimics reversed the increased expression of miR-374a-5p and the decreased expression of SM22α and α-SMA in PDGF-BB-induced VSMCs (Fig. 1E,F).These results indicated that miR-374a-5p was upregulated in IA patients and promoted PDGF-BB-induced VSMC phenotypic transformation.

In vivo validation of miR-374a-5p affecting vascular remodeling in IA rats
Next, we utilized an elastase-induced IA rat model to further investigate the effect of miR-374a-5p on vascular remodeling in IA rats.As shown in Fig. 2A,B, IA rats exhibited a significant increase in SBP and DBP, which was partially alleviated upon transfected with miR-374a-5p inhibitor.HE staining showed that the IA group had a disrupted endothelial layer, decreased VSMC count and layer number, and inflammatory cell infiltration compared to the Sham group.Still, these symptoms were improved in the IA + miR-374a-5p inhibitor group (Fig. 2C and Fig. S3).qRT-PCR results indicated the expression of miR-374a-5p was successfully suppressed in vascular wall tissue and plasma of IA rats (Fig. 2D,E).FISH-IF results showed that miR-374a-5b and SM22-α were co-localized in aneurysm vessels.The expression of miR-374a-5p was up-regulated in IA rats, which was significantly decreased after the application of miR-374a-5p inhibitor (Fig. 2F).In addition, compared to the Sham group, expression levels of SM22α, α-SMA, Calponin, TIMP3, and TIMP4 decreased, while MMP1, MMP3, and MMP9 increased in the serum of IA rats.However, miR-374a-5p inhibitor reversed these indicators (Fig. 2G).
Vol:.( 1234567890 Moreover, the IA group had a higher M1/M2 ratio than the Sham group, while the IA + miR-374a-5p inhibitor group showed opposite trends to those observed in the IA group (Fig. 2H). Figure 2I illustrated that, in IA rats, serum levels of IL-1β, IL-6, and TNF-α were observed to rise significantly in comparison to the Sham group.On the other hand, the levels of IL-10 dropped correspondingly.However, by suppressing miR-374a-5p, these changes were reversed.The above data suggested that miR-374a-5p influenced vascular remodeling in IA rats.

miR-374a-5p targeted WNT signaling
Using the TargetScan database, we anticipated the potential targeting impact of miR-374a-5p on the WNT signaling pathway.Subsequently, qRT-PCR and WB analysis revealed that the mRNA and protein expression levels of WNT2B, WNT3, and WNT5A happened to be markedly lower in IA rats when compared to the Sham group (Fig. 3A,B).Pearson analysis of the correlation between miR-374a-5p and WNT signaling (WNT2B, WNT3, and WNT5A) in IA rats was shown in Fig. 3C, and the correlation with WNT5A was the greatest, thus WNT5A was selected for further study.We also found that miR-374a-5p inhibitor upregulated the expression of WNT5A in IA rats (Fig. 3D).As Fig. 3E illustrated, the bioinformatics predicted that miR-374a-5p targeted WNT5A.Further reinforced by dual-luciferase reporter assays, it was discovered that miR-374a-5p mimics reduced WT-WNT5A luciferase activity when compared to miR-128-3p NC, while MUT-WNT5A had no significant difference.This strengthened the targeting relationship between miR-374a-5p and WNT5A (Fig. 3F).Overall, these results signified that WNT5A signaling was targeted by miR-374a-5p.

WNT5A-mediated miR-374a-5p affected VSMC phenotypic transformation
To investigate whether miR-374a-5p affects VSMC phenotypic transformation through WNT5A, we performed Box5 intervention on the cells.First, the expression of WNT5A was detected by WB.Significantly, we noticed that relative to the inhibitor NC group, WNT5A expression exhibited a significant increase in the miR-374a-5p inhibitor group.In contrast, WNT5A expression was reduced following co-treatment with Box5, as per the data displayed in Fig. 4A.Further, the IF analysis showed that miR-374a-5p inhibitor promoted SM22α and α-SMA expression, and partially rescued the effect of miR-374a-5p when co-treated with Box5 (Fig. 4B).The WB and ELISA findings indicated that Box5 treatment led to a reversal concerning the upregulation of SM22α, α-SMA, Calponin, TIMP3, and TIMP4 expression, and IL-10 levels.It also led to the downregulation of MMP1, MMP3, and MMP9 expression, and IL-1β, IL-6, and TNF-α levels induced by miR-374a-5p inhibitor (Fig. 4C,D).These outcomes suggested a mediation of WNT5A for the effects induced by miR-374a-5p on VSMC phenotypic transformation.Finally, we investigated the effects of WNT5A on THP-1 cell-derived macrophage polarization.We first measured the level of WNT5A in VSMCs using ELISA.The data in Fig. 5A indicated that WNT5A expression levels decreased significantly following treatment with Box5 compared to the miR-374a-5p inhibitor group, where they exhibited a rise.Next, THP-1 cells were co-cultured with PDGF-BB-treated VSMC cells, and flow cytometry analysis showed that the M1/M2 ratio was significantly decreased in the THP-1 + miR-374a-5p inhibitor group compared to the inhibitor NC group, and Box5 reversed the effects of miR-374a-5p inhibitor (Fig. 5B,C).These results suggested that miR-374a-5p affected M1 macrophage polarization through the WNT5A signaling.

Discussion
Given the detrimental consequences of subarachnoid hemorrhage due to IA rupture, preventative interventions to impede lesion rupture are essential for public health.Previous studies have shown an increased expression of miR-374a-5p in IA, yet its specific role and mechanisms remain elusive.In our study, miR-374a-5p was upregulated in IA patients.Importantly, miR-374a-5p was found to regulate VSMC phenotypic transformation and M1 macrophage polarization by targeting WNT5A, thereby impacting the progression of IA.Our findings contributed novel insight into the specific mechanism by which miR-374a-5p impacts the progression of IA via WNT5A signaling, signifying the innovative nature of our study.VSMCs are highly specialized contractile cells with considerable plasticity, displaying marked phenotype changes upon stimulation 25,26 .It's worth noting that VSMC phenotypic transformation in IA pathogenesis plays a crucial role in IA formation, development, and rupture 27 .Additionally, Macrophages are key effector cells in the progression of IA 28 .In IA development, there is increased polarization towards the M1 phenotype, and limiting macrophage M1 polarization may prevent IA formation and rupture 5,29 .Specifically, inflammatory cytokines secreted by M1 cells trigger pathological changes in the IA wall, such as TNF-α, which, on the one hand, initiates VSMC phenotypic transformation from contractile to pro-inflammatory and matrix remodeling phenotypes.On the other hand, stimulated VSMCs propagate the inflammatory cascade by secreting cytokines 30 , ultimately driving macrophage M1 polarization.In summary, the interaction between M1 macrophages and VSMCs may exacerbate the progression of IA through a positive feedback loop.Our study has revealed that SM22α, α-SMA, Calponin, TIMP3, TIMP4, and IL-10 levels were reduced in PDGF-BB-induced VSMCs, while MMP1, MMP3, MMP9, IL-1β, IL-6, and TNF-α levels were elevated.Notably, a similar phenomenon was observed in IA rats.Furthermore, IA rats exhibited a higher M1/M2 ratio, SBP, and DBP than normal rats.Additionally, IA rats presented with significant vascular lesions.These findings suggested that VSMC phenotypic transformation and macrophage polarization play important roles in the occurrence and development of IA.Presently, many studies are focusing on the impact of miRNAs in the creation and progression of IA.There is evidence to suggest that miRNAs contribute to the regulation of VSMC proliferation, migration, and apoptosis.For instance, Hou et al. reported the inhibitory effect of miRNA-370-3p on VSMC proliferation by targeting the KDR/AKT signal pathway and its role in IA occurrence and development 31 .In a similar vein, abnormal expression of miR-29b and miRNA-9 promotes the occurrence and rupture of IA by mediating VSMC phenotypes and inhibiting their proliferation 32,33 .These studies provide a basis for profound insights into pathology, and the exploration of potential clinical applications that could facilitate the prevention and treatment of IA.Recent studies have shown that miR-374a-5p is highly upregulated in the plasma of IA patients 12 .However, the specific mechanism underlying the role of miR-374a-5p in IA has not been fully illuminated yet and requires further investigation.Our study further confirmed that miR-374a-5p was overexpressed in the arteries of IA patients, mainly expressed in VSMCs.In vitro, knocking down miR-374a-5p reversed the promotion of SM22α and α-SMA expression by PDGF-BB, while overexpression of miR-374a-5p has the opposite effect.Meanwhile, downregulating miR-374a-5p reversed the decrease in Calponin, TIMP3, TIMP4, and IL-10 levels caused by PDGF-BB, and further reduced the levels of MMP1, MMP3, MMP9, IL-1β, IL-6, and TNF-α.These data were further validated in vivo, suggesting that miR-374a-5p regulated PDGF-BB-induced phenotypic transformation of VSMCs.Furthermore, in animal experiments, IA rats exhibited a significant increase in SBP and DBP, accompanied by vascular lesions, which was alleviated by miR-374a-5p downregulation.These findings indicated that miR-374a-5p impacted vascular remodeling in IA rats.
WNT5A is a representative WNT ligand that regulates cellular functions via the non-canonical WNT5A pathway 34 .Prior studies have indicated that WNT5A may participate in the pathogenesis of vascular disease by regulating the behavior of macrophages and VSMCs 16 .However, little is currently understood regarding the role of WNT5A signaling in the pathophysiology of IA.In this research, combining bioinformatics analysis and experimental validations, we found that miR-374a-5p targeted and negatively regulated WNT5A.miR-374a-5p inhibitor led to an increase in the expression of SM22α, α-SMA, Calponin, TIMP3, and TIMP4 as well as IL-10 content, and a decrease in the expression of MMP1, MMP3, and MMP9 and the content of IL-1β, IL-6, and TNF-α.The co-administration of Box5 reversed the effects of miR-374a-5p inhibitor.These results suggested that WNT5A mediated the effects of miR-374a-5p on the phenotypic transformation of VSMCs.Additionally, miR-374a-5p inhibitor led to a significantly decrease in M1/M2 ratio, which was reversed by Box5 the effects of.The above results indicated that miR-374a-5p affected M1 macrophage polarization via the WNT5A signaling.
In this study, we discovered that downregulating miR-374a-5p promoted VSMC phenotypic transformation to the contractile phenotype and inhibited M1 macrophage polarization, an effect reversed by the WNT5A antagonist Box5.Nevertheless, our research still has many limitations.Firstly, the clinical sample size of this study is limited, and larger sample sizes can be used for future verification.Secondly, further in-depth studies are needed to explore the mechanism of how PEGF-BB and IA models induce miR-374a-5p overexpression.Additionally, future research can further examine the expression of miR-374a-5p in other cell types, such as aortic endothelial cells, to reveal its role and regulatory mechanisms in different cell types.Furthermore, additional studies and clinical trials are needed to determine whether reducing BP can reduce the severity or mortality of IA.Overall, our results provide new evidence that miR-374a-5p regulated VSMC phenotypic transformation and M1 macrophage polarization via WNT5A, thus affecting the progression of IA.These results provide new insights into the development of new drugs for the treatment of IA, with the miR-374a-5p/WNT5A axis offering promise as a potential target for IA treatment, providing a fresh direction for future drug research and development. https://doi.org/10.1038/s41598-024-51243-zwww.nature.com/scientificreports/